Biochemistry I Sheet #35 Copy PDF

35 34 Lajneh Teejan Lajneh Teejan Lajneh Wisdom Lajneh Teejan + Rawan Asrawi Nafiz Abutarboush Nafez Abotarboush ❖NON- SPECIFIC REGULATORS (They don’t work against specific enzyme) ▪ RE...

35 34 Lajneh Teejan Lajneh Teejan Lajneh Wisdom Lajneh Teejan + Rawan Asrawi Nafiz Abutarboush Nafez Abotarboush ❖NON- SPECIFIC REGULATORS (They don’t work against specific enzyme) ▪ REGULATION THROUGH CHANGES IN AMOUNT OF ENZYME: They affect all enzymes in the same way because they deal with them as proteins. It works in an adaptive way so it needs time. o Regulated enzyme synthesis: - Regulated by increasing or decreasing the rate of gene transcription (induction & repression). - Usually slow in humans (hours to days). - Sometimes through stabilization of the messenger RNA. o Regulated protein degradation: - Can be degraded with a characteristic half-life within lysosomes. - Example:During fasting or infective stress: gluconeogenesis increase & synthesis of antibodies (protein degradation increases). - Degradation can be Increased by synthesis of ubiquitin(which is protein induce the degradation of enzymes). ▪ EFFECT OF TEMPERATURE: - Increase in T° increases the rate until the reaction reaches a max (≈50°): the optimal temperature of each enzyme is its denaturation temperature and when it denatures it loses its structure leading to the loss of the function. - Examples: 1-Autoclave steam heating. 2- Hypothermia, metabolic reactions, cardiac surgery. * We use this characteristic of enzyme in real life, such as in cardiac surgery but how? During cardiac surgery, tissue blood flow decreases due to the intentional lowering of the patient's body temperature. Freezing the patient slows enzymatic activity, subsequently reducing metabolism and the tissue's demand for blood. (We have to restore the body normal temperature in max 40 min or the patient may die)  Basic pH, decrease or It works at low pH ▪ EFFECT OF PH: increase in the pH the enzyme will be denature. - Each enzyme in the body has it's optimal PH value. - Most enzymes have their max. activity between (5-9). - Extremes of pH denatures protein (the salt bridges are affected) -pH can alter binding of substrate to enzyme (KM) by altering the protonation state of the substrate or altering Basic pH, increasing the pH doesn’t affect the The change in pH doesn’t affect it, it enzyme but decreasing doesn’t have a lot of the conformation of the enzyme. it has a huge effect. salt bridges. - The effect of pH is enzyme-dependent. ▪ EXTREMOZYMES: -Enzymes that exist in microorganisms that live in extreme environments and were used in industry. - Thermophiles (heat lovers). - Psychrophiles (cold lovers). -Examples: 1-Taq polymerase and PCR (for very high temp.) 2- Biobleaching of paper pulp using heat-stable xylanases (for very low pH value) 3-lipases and proteases (for very law temp.) ▪ ABZYMES-CUTTING EDGE SCIENCE: - An antibody that is produced against a transition-state analog (active) (very high affinity) - An abzyme is an antibody that works as enzyme for specific substrate, it is created in animals. - They are much more potent than regular enzymes. ▪ RIBOZYMES: - An exception to protein enzymes, ribozymes simply is RNA molecules that work as enzyme. - Examples: telomerase & RNase P, they catalyze their own reaction (any thing has RNA) - Catalyze splicing reactions and are involved in protein synthesis. - The catalytic efficiency of catalytic RNAs is less than that of protein enzymes but can greatly be enhanced by the presence of protein subunits. - They are very weak in their nature, they become stronger when they bind to proteins. ❖REGULATION OF METABOLIC PAYHWAYS - We talked about the regulation of enzymes either by phosphorylation, addition of a covalent group or make isozymes or other methods. On the other hand, in our bodies we don’t have a single reaction, we have series of reactions are called pathways. → PRINCIPLES OF PATHWAY REGULATION: 1. COUNTERREGULATION OF OPPOSING PATHWAYS - Synthesis vs. degradation (a different regulatory enzyme) -The principle is that if we have an enzyme induce synthesis of product for example, definitely the degradation process of this product must be reduced or stopped, so the synthesis and degradation pathways never happened at the same time. 2. TISSUE ISOZYMES OF REGULATORY PROTEINS - Accordingly, tissue work in different effectiveness 3. REGULATION AT THE RATE-LIMITING STEP Rate- limiting step - Pathways are principally regulated at their rate-limiting step (which is the step that determine the rate for all pathway) - The slowest step (the rate determining step) & is usually not readily reversible - Changes in this step can influence flux through the rest of the Pathway. - Usually the first committed step in a pathway. - Requirement for high amount of energy (in this way the body distinguishes it) - High KM values of enzyme towards its substrate (which means it needs a high concentration of substrate). 4. The committed step -A committed step in a metabolic pathway is the first irreversible reaction that is unique to a pathway and that, once occurs, leads to the formation of the final substrate with no point of return. - Committed steps are exergonic reaction. pathway T - For example look at the pathway T, the committed step for making product E is (B → C), not (A → B). - If you understand the concept, can you tell me what is the committed step for making the product Z from A? The answer isn’t B to X, because it reversible and you can back from X to B, and then you can make the product E. The correct answer is X to Y, because it is the first irreversible step which is unique for producing Z. Note: the committed step is usually the rate determining step (not always). 5. FEEDBACK REGULATION - This type of regulation is much slower to respond to changing conditions than allosteric regulation Negative feedback regulation (feedback inhibition) - When the concentration of the final product increases, it will come back and inhibit the enzyme that catalyzed the first step. Positive feedback regulation - The final product comes back and activates the enzyme that catalyzes the first step. (like the feedback for oxytocin during labor) Feed-forward regulation - When the first intermediate is produced it will go to activate the last enzyme in the pathway. So, once the substrate for the last enzyme is produced it will become a product. This pathway is used for disposal of toxins. 6. Enzyme compartmentalization - Both enzymes and their substrates are present in relatively small amount in a cell - A mechanism by which rate of reactions become faster is their compartment- alization; reducing area of diffusion - In this way, enzymes are sequestered inside compartments where access to their substrates is limited - Lysosomes; proteins get transported to lysozymes - Mitochondria; energy metabolic pathways Ex: Metabolism of fatty acids; synthesis (cytosol) vs. degradation (mitochondria) 7. Enzyme complexing (A multienzyme complex) - Complexing various enzymes that share one process - Product of enzyme A pass directly to enzyme B - Pyruvate dehydrogenase (mitochondria) 3 enzymes: decarboxylatin1, oxidation2, & transfer of the resultant acyl group to CoA3 ❖ ENZYMES IN MEDICAL DIAGNOSIS - The idea is that enzymes are not distributed equally in our body, and some of enzymes are found exclusively in some organs. - When you take a blood sample, and check the plasma, if it contains a higher concentration of one enzyme than normal, this indicates that cells which are supposed to contain this enzyme are degraded, so the enzyme became free in the blood. ▪ DIAGNOTIC ENZYMES & LIVER DISEASE - Examples of enzymes in liver: ALT, AST, LDH, CK (CPK) - In liver disease we care about : ALT (alanine transaminase) & AST (aspartate transaminase), ALT is the most specific for liver. - Ratio can also be diagnostic (ALT/AST) as: 1- In liver disease or damage (not of viral origin): ratio is less than 1(High AST) 2- With viral hepatitis: ratio will be greater than 1 (because the degradation with viral cause will be huge), (High ALT) - AST is found in heart and liver. In the liver, its concentration is higher than ALT but it isn’t specific as ALT, because again you find it in the heart. - ALT is found in tissues but in very low concentrations, so you can consider it exclusive for liver. ▪ MYOCARDIAL INFRACTION - When a patient come with chest pain the first thing you will do for them is ECG test and normal ECG doesn’t rule out myocardial infraction. So, you have to check the enzymes. 1- LDH (LACTATE DEHYDROGENASE) - LDH-1/LDH-2 ratio is diagnostic for myocardial infarction (heart attacks) - Normally, this ratio is less than 1 - Following an acute myocardial infarct, the LDH ratio will be more than 1 That’s mean we have Normally LDH-1 is myocardial infraction less than LDH-2 → the heart cells are dying, and their contents are in the blood. -LDH has 5 different isozymes. - LDH1 is found in the heart and RBCs. LDH5 are found in skeletal muscles. - In abnormal condition, LDH1 reach its peak after 72 hours. 2- CPK - In heart, skeletal muscles, & brain - Like LDH, there are tissue-specific isozymes of CPK: - CPK3 (CPK-MM): the predominant isozyme in muscle. - CPK2 (CPK-MB): accounts for ≈35% of CPK activity in cardiac muscle, but less than 5% in skeletal muscle - CPK1 (CPK-BB) is the characteristic isozyme in brain and is in significant amounts in smooth muscle - The CPK2 is the most common test for myocardial infraction nowadays.  CPK AND MYOCARDIAL INFARCTION - Most of released CPK after MI is CPK-MB. - Increased ratio of (CPK-MB/total CPK) may diagnose acute infarction, but an increase of total CPK in itself may not. - The CPK-MB is also useful for diagnosis of reinfarction because it begins to fall after a day and disappears in 1 to 3 days, so subsequent elevations are indicative of another event. ‫ بالتالي منه بقدر‬،‫ أيام‬٣-١ ‫ في حالة الجلطات القلبية بعلى تركيزه بالدم وبرجع يقل خالل‬CPK-MB ‫(يعني‬ ) ‫ شوفوا المثال‬..‫ اذا هاي الجلطة تكررت وال ال‬-٢ ، ‫ وجود جلطة‬-١ : ‫اشخص شغلتين اذا شفته عالي‬ * If the patient comes to the hospital and they suffer from chest pain from 4 days, and you check his CK-MB and you find it high, what is the case here? It is a reinfarction after the first infraction. ‫ وجع‬،)‫ أيام (طبعًا احسن مكان يجيه هو عيادة االسنان تبعتك‬٤ ‫اذا اجا مريض بوجع في الصدر من‬- ‫ بتعمله فحوصات لهاالنزيم اذا كانت نسبته عالية بالدم بتتأكد من‬.. ‫الصدر بخليك تشك في جلطة قلبية‬ ٤ ‫ وبما انه حكينا هاالنزيم برتفع وبرجع يختفي خالل يوم او يومين والمريض عنده الوجع من‬،‫الجلطة‬ ‫أيام اذًا اكيد ارتفع اكتر من مرة خالل هالفترة يعني صار اكتر من جلطة وحدة على مدى هاالربع أيام‬ Reinfarction : ‫وهالحالة الي بنسميها‬ → Notice the curve of CK-MB , increase then decrease in about 24 hours (fastly).  **‫**هاي الصفحة قراءة بفهم ال تحفظوا القيم بس افهموا كيف ممكن تكون‬ - Sample #3 represents results for a control. - Sample #8 results are from a normal specimen. - Sample# 1 MI patient. The specimen was collected at a time when the activity of both LDH and CK were elevated. Note the LDH flip and the high relative activity of the MB isoenzyme. - Sample# 2 MI patient who experienced chest pain only several hours previously. Total CK is significantly elevated with a high relative MB isoenzyme activity. - Sample# 6 MI patient (the 1st day post MI); CK activity is definitely elevated with a high relative MB isoenzyme activity and the LDH flip is evident. - Sample# 5 MI patient (2 days post MI) so that CK has almost returned to normal activity and the LDH flip is definite. - Sample# 7 MI patient with complications of heart failure and passive liver congestion or the patient was involved in an accident as a consequence of the MI, and suffered a crushing muscle injury. - Sample# 4 a patient with liver disease. Although the LDH isoenzyme pattern is indistinguishable from muscle disease or injury, the absence of at least a trace of CK- MB isoenzyme is inconsistent with the muscle CPK isoenzyme distribution as is the apparently normal total activity. THE END OF SHEET #34